Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants  Manabu Ote, Morio Ueyama, Daisuke Yamamoto  Current Biology  Volume 26, Issue 17, Pages 2223-2232 (September 2016) DOI: 10.1016/j.cub.2016.06.054 Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Structure of the WD1278 Gene and Its Evolutionary Diversification among the Wolbachia Groups (A) The WD1278 gene is duplicated in the genome of a group B Wolbachia (wPip) and degenerates in the genome of a group D Wolbachia (wBm). While absent in the wMel homolog, ankyrin repeats are present in the group B homolog. The region isolated from the screening (82–447 amino acid [aa] residues) is indicated with a horizontal arrow. (B) The WD1278 homologs found in group A Wolbachia strains are split into two genes in wRi and wAna, while the 3′ half is absent in wHa and wBif. (C and D) The full-length WD1278 impairs female reproduction. The number of eggs laid in 3 days (C) and hatching rates (D) by females expressing 1–533 aa residues containing the homologous region between groups A and B Wolbachia (WD12781–533), 1–839 aa residues excluding the C-terminal hydrophobic regions (WD12781–839), or the full-length (WD12781–921) are shown. All proteins were expressed as a fusion with mRFP. The codons of the WD1278 genes were optimized for expression in Drosophila. Mean ± SEM values are shown. The statistical significance from the control was evaluated by the Tukey-Kramer honestly test (∗p < 0.01). n, the number of females examined. See also Figures S1 and S2. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Localization of Endogenous TomO (A and B) Bacterial TomO was detected in ovaries only when infected with Wolbachia. The anti-TomO immunoreactive material colocalized with Wolbachia in an infected germarium (A and A’), whereas it was not detected in an uninfected germarium (B and B’). Staining the germaria with an antibody that recognizes the prokaryote-specific FtsZ protein revealed the presence or absence of Wolbachia (A” and B”). Scale bar, 5 μm. (C) The portion of an embryo examined for the localization of Wolbachia and TomO in (D)–(G’) is indicated with a square on the schematic drawing of an embryo. Wolbachia (magenta) are known to accumulate at the posterior pole. (D and E) The posterior localization of Wolbachia (D) and TomO (E) was revealed by staining paraformaldehyde-fixed embryos with the anti-FtsZ and anti-TomO antibodies, respectively. (F and G) Staining with the anti-FtsZ (F and F’) and anti-TomO (G and G’) antibodies microinjected into embryos prior to paraformaldehyde fixation. Note that the antibodies could not penetrate the bacterial wall in unfixed embryos. The anti-FtsZ antibody failed to react with the antigen FtsZ within the bacteria (F and F’), whereas the anti-TomO antibody successfully reacted with the antigen TomO (G and G’), presumably because TomO was secreted from the bacteria into the embryonic cytoplasm. Dashed lines represent the posterior contours of embryos. Scale bar, 5 μm. See also Figure S3. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 TomO Overexpression as well as Wolbachia Infection Restore GSCs in an Sxlf4 Mutant Germarium (A–C) Germaria of heterozygous (A) and homozygous (B and C) Sxlf4 females without (A and B) and with (C) Wolbachia infection. Wolbachia were detected by the anti-FtsZ antibody (A’–C’). Green, pMad; magenta, Vas. Scale bar, 10 μm. (D) Quantification of GSC numbers under the conditions shown in (A)–(C) and (E)–(G). The mean ± SEM values are shown. Statistical significance was evaluated by the Tukey-Kramer honestly test (∗p < 0.01; N.S., not significant [p > 0.05]). n indicates the number of germaria examined, which were derived from at least 12 females. (E–G) Germalia of Sxlf4 homozygous females carrying UAS-mRFP (E), UAS-mRFP::TomOΔHS (F), or UAS-mRFP::TomOFL (G). Germ cells were labeled by the anti-Vasa antibody (magenta) and GSCs by the anti-pMad antibody (green). Scale bar, 10 μm. (H–J) pMad immunoreactivity in germline mosaic clones expressing control mRFP (H), mRFP::TomOΔHS (I), or mRFP::TomOFL (J). The genotypes of the flies are Sxlf4 hsFLP; +/UASp-mRFP or mRFP-TomO; FRT82B Ubi-GFP nos-GAL4::VP16/FRT82B. Arrows indicate GSCs expressing the transgene. Green, pMad; magenta, RFP. Scale bar, 10 μm. (K) Quantification of fluorescence intensities of pMad in GSCs under the three conditions shown in (H)–(J). The mean intensity of fluorescent signals per pixel in the nucleus of single GSC expressing the transgene was compared to that of control GSC not expressing it in the same germarium. The mean ± SEM values are shown. Statistical significance was evaluated by the Student’s t test compared to the control expressing mRFP (∗p < 0.001; N.S., not significant, p > 0.05). n indicates the number of germaria examined, which were derived from at least 16 females. Scale bar, 10 μm. See also Figures S4 and S5. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 TomO Interacts with nos mRNA (A–C) Overexpressed TomO (A, TomOΔHS; B, TomOFL) as well as infecting Wolbachia (C) were localized in particles that contain Cup in nurse cells. The tissue was triply stained with DAPI for DNA labeling (blue), the anti-Cup (green), and the anti-RFP antibody (red). Insets in (A)–(B”) are enlarged images of the boxed regions, in which colocalization of TomO and Cup is evident. Arrows in (C)–(C”) indicate colocalization of Wolbachia and Cup. Wolbachia were detected by the anti-FtsZ antibody (C’). Scale bar, 10 μm. (D) Western blot analysis of immunoprecipitates of lysates prepared from ovaries that express an indicated transgene. The anti-RFP antibody was used for immunoprecipitation of mRFP, mRFP::TomOΔHS, and mRFP::TomOFL. (E) Immunoprecipitation conducted as in (D) enriched nos mRNA significantly more than rp49 mRNA, a control. The mean ± SEM values of six replicates are shown. The statistical significance was estimated between the value for mRFP precipitate and that for mRFP-TomOΔHS or mRFP-TomOFL precipitate by the Student’s t test (∗p < 0.05; N.S., not significant). See also Figures S3 and S6. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 Endogenous TomO Colocalizes with nos mRNA (A) The posterior pole of a Wolbachia-infected embryo (cf. Figure 2C) triply stained for DNA of primarily bacterial origin (blue), bacterial TomO (green), and nos mRNA of host origin (red) in (A). Images stained for TomO and nos mRNA also are shown in black and white in (A’) and (A”), respectively. Scale bar, 3 μm. (B and C) Associations of TomO with nos mRNA revealed by the PLA. TomO was detected by an anti-TomO antibody, whereas the DIG-labeled antisense probe for nos mRNA was detected by an anti-DIG antibody. The secondary antibody for the detection of the anti-TomO antibody carried a DNA probe “A” for PLA, whereas the secondary antibody for the detection of anti-DIG antibody carried a complementary DNA probe “B” for PLA. The secondary antibody emits fluorescence only when two probes, A and B, hybridize to each other, signifying the close proximity between two antigens, i.e., TomO and nos mRNA. The PLA yielded fluorescence in the Wolbachia-infected (B’) embryo, but not in the uninfected embryo (C’). No PLA signal was detected with a control sense probe as shown in Figure S3C. Scale bar, 3 μm. Dashed lines represent the posterior contours of embryos. See also Figure S3 and Table S1. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 TomO Promotes Nos Expression (A–C) Nos expression as detected with a nos promoter-driven EGFP::nos transgene (green) in germline clones that carry UAS-mRFP (A–A”), UAS-mRFP::TomOΔHS (B–B”), or UAS-mRFP::TomOFL (C–C”) together with nos-GAL4. Because nos-GAL4 was genetically linked with Ubi-mRFP-nls, the absence of mRFP (red) signified the absence of expression of UAS transgenes. DNA was labeled with DAPI (blue). Arrows indicate the clonal tissues in which the indicated transgene was expressed. The genotype of flies used to generate TomO mosaics was Sxlf4 EGFP::nos/Sxlf4 hsFLP; + / UASp-mRFP::TomO; FRT82B Ubi-mRFP-nls nos-GAL4::VP16/FRT82B. Scale bar, 10 μm. (D) Quantitative comparisons of fluorescence intensity for EGFP-Nos among GSCs that express mRFP, mRFP::TomOΔHS, or mRFP::TomOFL. The fluorescence intensity in single GSC expressing the transgene was compared to that of control GSC not expressing it in the same germarium. The mean ± SEM are shown. Statistical significance was evaluated by the Student’s t test compared to the control expressing mRFP (∗p < 0.05; N.S., not significant). n indicates the number of germaria examined, which were derived from at least 16 females. See also Figure S7. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 7 Rescue of GSCs in an Sxlf4 Mutant Germarium by Wolbachia Requires Functional Nos (A–D) Nos expression (A–C), but not Vasa expression (A’, B’, and D), in Sxlf4 mutant GSCs was increased by Wolbachia infection. GSCs are indicated by arrows. Nos expression was monitored by EGFP-Nos. Wolbachia were detcted by the anti-FtsZ antibody (A” and B”). The genotype of the flies is Sxlf4/Sxlf4 egfp-nos+; +/nosRC. nosRC is a loss-of-function allele of nos. Scale bar, 10 μm. (C and D) Quantifications of fluorescence intensity of Nos (C) or Vasa (D) in Sxlf4 mutant GSCs with or without Wolbachia infection are shown. The mean ± SEM are shown. Statistical significance was evaluated by the Student’s t test (∗p < 0.005; N.S., not significant, p > 0.05). n indicates the number of germaria examined, which were derived from at least eight females. (E) The number of pMad-positive GSCs was increased by introducing two additional copies of nos into Sxlf4 homozygotes. The mean ± SEM are shown. The statistical significance was evaluated by the Student’s t test (∗p < 0.05). n indicates the number of germaria examined, which were derived from at least 12 females. (F–H) Wolbachia infection failed to increase the number of pMad-positive cells in Sxlf4 homozygous females that were also mutant for nos (nosRC/Df). Wolbachia were detected by the anti-FtsZ antibody (F’ and G’). The mean ± SEM are shown. The statistical significance was evaluated by the Student’s t test (N.S., not significant, p > 0.05). GSCs are indicated by arrows. Scale bar, 10 μm. n indicates the number of germaria examined, which were derived from at least 12 females. Current Biology 2016 26, 2223-2232DOI: (10.1016/j.cub.2016.06.054) Copyright © 2016 Elsevier Ltd Terms and Conditions